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Amniotic fluid cell chromatin examination

For families with a family history of X-linked recessive hereditary diseases, the parental phenotype is normal. If the mother is a carrier of the disease-causing gene, the probability of the son of the child is 50%; the daughter has a normal phenotype, but half of it. May be a carrier. When the father is a patient, the sons are all normal, and the daughters are all carriers of amniotic fluid cell chromatin examination to predict the sex of the fetus to estimate the probability of occurrence of certain hereditary diseases.

Positive: The probability of X-linked recessive recessive disease is determined based on the results.

Tips: Amniocentesis is usually performed in the second trimester (16-21 weeks of gestation). Normal value

X chromatin > 0.06 for female fetuses, <0.05 for male fetuses;

Y chromatin > 0.05 is a male fetus and < 0.04 is a female fetus.

Clinical significance

For families with a family history of X-linked recessive hereditary diseases, the parental phenotype is normal. If the mother is a carrier of the disease-causing gene, the probability of the son of the child is 50%; the daughter has a normal phenotype, but half of it. May be a carrier. When the father is a patient, the son is all normal, and the daughter is all carriers, such as hemophilia A, hemophilia B, glucose-6-phosphate dehydrogenase deficiency, red-green blindness, pseudohypertrophic muscular dystrophy Congenital gamma globulin deficiency.

Amniocentesis is usually performed in the second trimester (16-21 weeks of gestation). The urine should be drained before surgery, with both hands on the hips and gently turn the waist and abdomen. Then supine, use B-ultrasound to detect the positioning, select the puncture point, and puncture under strict aseptic operation conditions. Generally, about 20 ml of amniotic fluid is taken and placed in a clean and sterilized centrifuge tube for immediate inspection.

Inspection process

(1) Take 15 to 30 ml of amniotic fluid for 16 to 20 weeks of pregnancy, and centrifuge at 1000 r/ml for 10 min.

(3) Transfer into a 25 ml square culture flask, add 3 ml of a medium having a pH of 6.5 to 6.8 (containing penicillin 100 U/ml, streptomycin 100 μg/ml) and 1 ml of calf serum, and incubate in a 37 ° C incubator.

(4) A large number of fibroblast-like or epithelioid cell colonies were observed after 7-10 days of culture. The fresh medium can be exchanged at this time.

(5) When the cell colonies are expanded into pieces and there are many translucent circular dividing cells, colchicine can be added to make the final concentration 0.1-0.3 μg/ml, and then cultured in a 37 ° C incubator for 5-6 h. (The above procedures are all carried out under aseptic conditions). Harvesting standards: 1 with fibroblasts as the main type, the growth of the cells; 2 with 10x eyepieces and 20x objective observation, the growing cell clones cover 1 or more complete fields of view; 3 see more than 10 translucent Round cells and more than 10 double round bright dividing cells.

(6) If the cells are not growing vigorously, the growth cycle is not uniform or the cells are aging, and the harvesting standard is not reached, the subculture can be continued in the original bottle. Method: Under sterile conditions, the culture solution was first poured, and a few drops of 2.5 g/L sterile trypsin solution were added, and the mixture was shaken and poured. Further, 5 to 10 drops of trypsin were added, and gently shaken at 37 ° C for about 5 minutes to cause the adherent cells to fall off. 4 ml of fresh medium containing calf serum was added, and the culture was allowed to stand at 37 ° C for 4 to 5 hours. Carefully change the culture medium and continue the culture. It can be harvested 3 to 5 days after the passage.

(7) Transfer the culture solution to a graduated centrifuge tube, add 1 ml of ED-TA-trypsin solution to the flask, place in a 37 ° C incubator for 5 min, and then rinse the adherent cells with an elbow pipette (2.5 g/ can also be used) L trypsin solution digestion). The cells detached from the bottle wall were poured into a centrifuge tube, mixed with the original culture liquid phase, and the culture flask was washed with a small amount of warm physiological saline, and the washed cells were also poured into the centrifuge tube and centrifuged at 1000 r/min for 10 min.

(2) Injury of the fetus, placenta and umbilical cord: The puncture needle can damage the fetus and can cause bleeding, and the placenta and umbilical cord can also cause bleeding or hematoma. Therefore, the source of bleeding should be identified when taking hemorrhagic amniotic fluid. If you suspect that you are from a fetus, you should continue to listen to the fetal heart.

(4) abortion or premature birth: the incidence of abortion or premature delivery is 0.1% -0.2%, often occurs within one week after surgery, even after the puncture, premature rupture of membranes leads to premature delivery.

(5) intrauterine infection: there may be maternal fever after surgery. Intrauterine infection can cause abnormal fetal development, or even fetal death. Therefore, amniocentesis should be strictly aseptic.